Project Summary A fundamental hallmark of human cancers is their ability to overcome replicative senescence and achieve cellular immortality. Normally silenced during differentiation in somatic cells, 90% of human tumors reactivate Telomerase Reverse Transcriptase (TERT) expression early during tumorigenesis to achieve cellular immortality. TERT, the catalytic subunit of telomerase, complexes with the RNA template molecule TERC and other proteins and binds to and extends the repetitive sequences at chromosomal ends, known as telomeres. Recently, non-coding mutations in the TERT promoter (TERTp) have been described in many cancers. These TERTp mutations are the most common non-coding mutation across all cancers and are the most frequent mutations in many cancers, such as 83% of IDH wildtype glioblastomas (GBM), the most common and deadly form of adult brain tumors. These mutations result in the formation of canonical E26 Transformation Specific (ETS) binding motifs that work in tandem with TERTp native ETS sites to recruit a specific ETS transcription factor, the GA-binding protein (GABP) complex. Our lab has demonstrated that this recruitment of GABP is necessary for TERT reactivation and maintenance of tumor cell immortality. Furthermore, our lab has shown, through CRISPR-cas9 mediated mutation of the tetramer forming subunit, GABPB1L (B1L), that the tetrameric form of GABP is necessary for this maintenance of tumor cell immortality. Interestingly, while CRISPR-Cas9 mediated ablation of B1L does not show a reduction in TERT expression, it does show a large increase in the GABP dimer specific subunit, GABPB1S (B1S). Importantly, it seems that this increase in B1S expression may be due to a GABP tetramer mediated negative feedback loop. Furthermore, preliminary evidence suggests that this increase in B1S may allow B1S containing dimers to bind to the mutant TERTp and maintain TERT expression. Broadly, this study aims to elucidate the molecular mechanisms underlying the maintenance of TERT expression during states of B1L elimination. In doing so, this study will determine the mechanisms underlying B1S expression upregulation during B1L reduction and will determine if B1S-containing GABP dimers are competent regulators of the mutant TERTp. Telomerase has long been an attractive therapeutic target for the reversal of tumor cell immortality; however, attempts to target telomerase have been mostly unsuccessful due to telomerase expressing stem cells. Prior studies suggest that targeting B1L in TERTp mutant cancers would allow for tumor specific inhibition of telomerase, however, our data have revealed that this approach is more complicated than previously appreciated. Importantly, the findings from these investigations will guide the design and development of effective and durable therapies targeting the GABP-TERT axis for the reversal of tumor cell immortality in TERTp mutant cancers such as glioblastoma, melanoma, bladder cancer, and many others.